Achieving tolerance remains the most important goal in transplantation immunology. Models of tolerance employing co-stimulatory blockade are among the most robust and clinically relevant approaches. Much is known about the mechanisms of tolerance that are operative during co-stimulatory blockade, such as anergy, apoptosis, and regulatory T cells (Treg). However, it is often difficult to induce and maintain robust tolerance that is resistant to external perturbations. This suggests that other important immunologic mechanisms that determine tolerance remain to be elucidated. Our laboratory has focused on the role of migration, trafficking and secondary lymphoid organ structure as crucial regulatory processes that determine whether immune interactions result in immunity versus tolerance. In several key publications we demonstrated that tolerance is initiated in lymph nodes (LN) through the precise interaction of specific alloantigen presenting cells with nave antigen specific T cells to generate regulatory suppressive T cells. This interaction occurs in the LN, and is dependent on the intricate coordination of many molecular signals. Subsequent trafficking of the suppressive T cells is critical, so that migration from blood to grafts and then into lymphatic has distinct and unique suppressive effects, in comparison to migration through blood and LNs. In the first funding period for this project, we elucidated several novel and unexpected mechanisms that are required for tolerance induction, and these mechanisms relate to the interaction of LN structure with the cellular and molecular mechanisms of lymphocyte responses. The results demonstrate that T cells destined to become suppressors are found in only one region of the LN, called the cortical ridge. In contrast, T cells destined to become effectors are found scattered throughout the LN. The cortical ridge is particularly rich in specialized fibers and stromal cells, called fibroblastic reticular cells (FRC), suggesting a uniqu function or arrangement for these cells and their associated fibers. These observations suggest the hypothesis that the FRC of the cortical ridge regulate critical aspects of LN structure and function, are key controlling elements for the positioning and function of T cells and antigen presenting cells, and therefore determine the choice between immunity versus tolerance.